Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, is critically required for murine embryonic development; animals lacking this caspase die around e10.5. This lethality cannot be attributed to a defect in apoptosis; leading to the idea that caspase-8 has an independent survival function. Recently we showed that development in caspase-8-deficient animals is fully rescued by ablation of RIPK3, a kinase that triggers a programmed form of necrosis (sometimes called necroptosis). In vitro studies showed that a complex of an adapter, FADD, caspase-8, and the caspase-8-like molecule FLIP, associates with RIPK3 (and another kinase, RIPK1), and the proteolytic activity of caspase-8-FLIP then prevents RIPK-dependent necrosis. Intriguingly, knockout of either FADD or FLIP causes the same embryonic lethality observed in caspase-8- deficient mice. Our central hypothesis, upon which this application is based, is that a complex of the adapter molecule, FADD, caspase-8, and FLIP, while not promoting apoptosis, antagonizes the formation of a necrosis-inducing RIPK1-RIPK3 complex, and thereby protects cells and animal development-this is the primary auxiliary (survival) role for FADD, FLIP, and caspase-8. Based on this hypothesis, we propose: 1. to formally test a model of FADD, FLIP, and caspase-8-mediated survival. Here, we will investigate the roles of FADD, FLIP, and caspase-8 in promoting and/or preventing apoptosis and RIPK-dependent lethality in development and in adult animals. Our model makes a number of predictions that will be tested in vivo and in vitro. 2. To investigate how the FADD-Caspase-8-FLIP complex antagonizes the RIPK1-RIPK3 complex function without inducing apoptosis. The catalytic activity of caspase-8 is required for cell survival. We will test three possibilities (which are not mutually exclusive): a. that caspase-8-FLIP has a different specificity than pro-apoptotic caspase-8 homodimers; b) that caspase-8-FLIP and caspase-8 homodimers have different access to substrates; and/or that caspase-8-FLIP heterodimers and caspase-8 homodimers differentially target caspase-8 and RIPK1-RIPK3 for degradation or inactivating modifications. We will perform our studies by utilizing potential substrates for caspase-8-FLIP and caspase-8 homodimers and determine if cleavage is required for protective effects. Our studies test these possibilities in vitro and in vivo.3. To delineate the mechanism(s) of RIPK-dependent necrosis. Using a novel system in which RIPK3 kinase activity induces necrosis independently of caspase or RIPK1 activity we will perform candidate and unbiased approaches to identify the mechanisms of this rapid, active necrosis. The finding that caspase-8-RIPK3 double knockout (DKO) mice are developmentally normal is a game changer that demands that the many proposed roles for FADD, caspase-8, and FLIP in cellular processes be rigorously re-examined with the possibility that all such effects are a direc outcome of the regulatory interactions between the FADD-caspase-8-FLIP complex and the RIPK1-RIPK3 function in triggering necrosis (and possibly other cellular effects of RIPK3). The experiments proposed in this application will provide such a re-evaluation, while providing deep insights into the mechanisms and regulation of RIPK-dependent necrosis.